Trimeric purine nucleoside phosphorylase: Exploring postulated one-third-of-the-sites binding in the transition state

Wielgus‐Kutrowska, Beata; Breer, Katarzyna; Hashimoto, Motomu; Hikishima, Sadao; Yokomatsu, Tsutomu; Narczyk, Marta; Dyzma, Alicja; Girstun, Agnieszka; Staroń, Krzysztof; Bzowska, Agnieszka

doi:10.1016/j.bmc.2012.08.045

Cited by 12 publications

(7 citation statements)

References 37 publications

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“…Recombinant E. coli and calf spleen PNP were obtained and purified as described elsewhere [31,32]. 2,6-Diamino-8-azapurine sulfate, N7-methylguanosine and 8-azaguanine were from Sigma-Aldrich (St. Louis, MO, USA.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Synthesis of Highly Fluorescent 8-Azapurine Ribosides Using a Purine Nucleoside Phosphorylase Reverse Reaction: Variable Ribosylation Sites

Stachelska-Wierzchowska¹,

Wierzchowski²,

Wielgus‐Kutrowska

et al. 2013

Molecules

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Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-β-D-riboside (λ max 365 nm), while for N8-β-D-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.

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Section: Methodsmentioning

confidence: 99%

Enzymatic Synthesis of Highly Fluorescent 8-Azapurine Ribosides Using a Purine Nucleoside Phosphorylase Reverse Reaction: Variable Ribosylation Sites

Stachelska-Wierzchowska¹,

Wierzchowski²,

Wielgus‐Kutrowska

et al. 2013

Molecules

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“…However, the subsequent work proved no negative cooperativity between subunits, neither in the ground, nor in the transition, state [7]. In addition, the trimeric PNP showed the stoichiometry of three ligand molecules, including the transition state analogue inhibitor, immucillin, bound per enzyme trimer [7]. This discrepancy was explained in our previous work [3,8].…”

Section: Introductionmentioning

confidence: 84%

“…According to [5,6], the trimeric PNP exhibits third-the-sites reactivity and one-third-of-the-sites binding in the transition state. However, the subsequent work proved no negative cooperativity between subunits, neither in the ground, nor in the transition, state [7]. In addition, the trimeric PNP showed the stoichiometry of three ligand molecules, including the transition state analogue inhibitor, immucillin, bound per enzyme trimer [7].…”

Section: Introductionmentioning

confidence: 90%

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

Dyzma

Wielgus‐Kutrowska

Girstun

et al. 2023

IJMS

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Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

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“…49) was prepared. 190 Although its activity was not better than the activity of the parent compound 205 (K i = 0.085 nM, Fig. 48) or immucilin H (209, K i = 0.023 nM, Fig.…”

Section: Anps With Fluorine Atom(s) At Phosphonate α-Carbonmentioning

confidence: 92%

“…In order to better understand the binding of the transition state analogues to the trimeric calf PNP, aza‐DFPP‐DG 210 ( K i = 6.8 nM, Fig. ) was prepared . Although its activity was not better than the activity of the parent compound 205 ( K i = 0.085 nM, Fig.…”

Section: Acyclic Nucleoside Phosphonatesmentioning

confidence: 99%

Medicinal Chemistry of Fluorinated Cyclic and Acyclic Nucleoside Phosphonates

Baszczyňski

Janeba

2013

Medicinal Research Reviews

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The fluorine atom plays an important role in medicinal chemistry because fluorine substitution has a strong impact on the physical, chemical, and biological properties of bioactive compounds. Such fluorine modifications have also been extensively studied among the pharmaceutically important class of nucleoside phosphonates, nucleotide analogues in which the phosphate group is replaced by the enzymatically and chemically stable phosphonate moiety. The fluorinated nucleoside phosphonates abound with antiviral, antiparasitic, and anticancer properties because they are able to act as inhibitors of important enzymes of nucleoside/nucleotide metabolism. In this paper, we review the biological properties of cyclic and acyclic nucleoside phosphonates modified by the attachment of one or more fluorine atoms to various parts of the molecule, namely to nucleobases, alkylphosphonate groups, cyclic or acyclic linkers, or to prodrug moieties.

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Trimeric purine nucleoside phosphorylase: Exploring postulated one-third-of-the-sites binding in the transition state

Cited by 12 publications

References 37 publications

Enzymatic Synthesis of Highly Fluorescent 8-Azapurine Ribosides Using a Purine Nucleoside Phosphorylase Reverse Reaction: Variable Ribosylation Sites

Enzymatic Synthesis of Highly Fluorescent 8-Azapurine Ribosides Using a Purine Nucleoside Phosphorylase Reverse Reaction: Variable Ribosylation Sites

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

Medicinal Chemistry of Fluorinated Cyclic and Acyclic Nucleoside Phosphonates

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